Vaccines comprising tb 10.4

ABSTRACT

Vaccination with the combination of Ag85B-TB10.4 and IC31® adjuvant generated a high amount of polyfunctional CD4 + T cells expressing high levels of IFN-γ, TNF-α, and IL-2. This in turn led to significant protection against infection with  M. tuberculosis  in the mouse aerosol challenge model of tuberculosis. Both the immunogenicity of the vaccine and its ability to protect against TB infection was highly dependent on the antigen dose. Thus, whereas the standard antigen dose of 5 μg, as well as 15 μg, did not induce significant protection against  M. tuberculosis,  reducing the dose to 0.5 μg increased both the immunogenicity of the vaccine as well as its protective efficacy to a level comparable to that observed in BCG vaccinated mice. Thus, the IC31® adjuvant, with the specified antigen dose, can induce a strong protective Th1 response against  M. tuberculosis.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit under 35 USC 119(e) of prior U.S.Provisional Patent Application No. 61/085,973, filed Aug. 4, 2008, whichis incorporated herein by reference.

BACKGROUND OF THE INVENTION

The global effort to develop a more effective Mycobacterium tuberculosis(M. tuberculosis) vaccine than the currently used Bacillus of Calmetteand Guerin (BCG) vaccine involves different strategies such as liveattenuated vaccines (Horwitz, et al., 2000), virally vectored M.tuberculosis vaccines (McShane, et al., 2004), and subunit vaccines(Olsen, et al., 2001 and Skeiky, et al., 2004). The subunit approachholds a number of advantages, such as increased safety and stability aswell as the demonstrated ability to boost prior BCG vaccination (Brandt,et al., 2004; Dietrich, et al., 2006). In addition, as subunit vaccinesappear not to be influenced by environmental mycobacteria, this type ofvaccine may be of particular use in the developing world (Brandt, etal., 2002). However, progress in this field has been delayed by the lackof adjuvants that induce a strong cell-mediated immune (CMI) response.Therefore, a need still remains for an immunogenic composition which cangenerate polyfunctional immune cells thereby providing greaterprotection against M. tuberculosis.

SUMMARY OF THE INVENTION

An immunogenic composition and vaccine for mammalian use with a low doseof an antigen comprising TB 10.4 fused to a polypeptide of the antigen85-complex (Ag85, composed of the Ag85A, Ag85B, and Ag85C proteins(Dietrich, et al., 2005)), e.g., Ag85B in an adjuvant, and methods ofimmunization and treatment of M. tuberculosis, are provided.

An immunogenic composition for mammalian use is provided comprising aTB10.4 protein and an Ag85-complex protein (described herein), whichoptionally can be fused together or provided as separate proteins,wherein the total amount of protein is less than about 25 μg, or lessthan 10 μg or equal to about 0.5 μg per antigen dose. In a furtherembodiment, the composition is for human use. In one embodiment, thecomposition does not contain dimethyl dioctadecyl ammonium bromide(DDA). An immunogenic composition described herein can additionallycomprise an adjuvant. In one embodiment, the adjuvant has at least onepolycationic peptide and at least one oligonucleotide, and in a furtherembodiment the oligonucleotide is a TLR9 (toll-like receptor 9) agonist.In one embodiment, the adjuvant is IC31® adjuvant (described herein). Ina further embodiment, the protein is from the Ag85-complex is an Ag85Bprotein.

In another embodiment, a vaccine is provided for mammalian use whichcomprises the above mentioned immunogenic composition. In a furtherembodiment, the vaccine is for human use. In still another embodiment, amethod of inducing protection against M. tuberculosis in a mammal isprovided, the method comprising introducing into the mammal animmunogenic composition as described above. In a further embodiment, themethod is for inducing protection in a human.

Still other aspects and embodiments of the invention will be apparentfrom the detailed description of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1D illustrate the concentration of IFN-γ released uponstimulation of peripheral blood mononuclear cells (PBMCs) with either 1or 5 μg/ml of Ag85B, TB10.4, control antigen CFP10, or no antigen.Stimulation is post-vaccination with varying doses of Ag85B-TB10.4(Hyvac4, i.e., H4) formulated in IC31® adjuvant—15 μg H4 (FIG. 1C), 5 μgH4 (FIG. 1B), 0.5 μg H4 (FIG. 1D), or control (FIG. 1A). Valuesrepresent the means of triplicate and SEMs are indicated by bars.

FIGS. 2A-2D illustrate IFN-γ released upon stimulation of PBMC's (FIG.2A)/(FIG. 2C) and splenocytes (FIG. 2B)/(FIG. 2D) with Ag85B (FIG.2A)/(FIG. 2B) or TB10.4 (FIG. 2C)/(FIG. 2D), post-vaccination with 0.5μg, 5 μg, or 15 μg or 0 μg (non-vacc.) of H4 formulated in IC31®adjuvant. In (FIG. 2A)-(FIG. 2D), a vaccination dose of 0.5 μg H4 gavesignificantly (p<0.05, one-way ANOVA and Tukey's post test) higherantigen responses, compared to vaccination doses of 5 μg and 15 μg.

FIGS. 3A and 3B illustrate the protective efficacy of H4. FIGS. 3A and3B reflect data obtained from two experiments (repeated). The groups ofmice reflected are non-vaccinated (negative control), Bacillus ofCalmette and Guerin (BCG) immunized (positive control), 0.5 μg H4(Ag85B-TB10.4 fusion protein in IC31® adjuvant), 5 μg H4, and 15 μg H4.Following challenge with aerosolized virulent M. tuberculosis (Erdmanstrain), colony forming units (CFU) in the lungs were determined. Log₁₀CFU is reflected for each group.

DETAILED DESCRIPTION OF THE INVENTION

The combination of Ag85B-TB10.4 (Hyvac 4) and the IC31® adjuvant as animmunogenic composition and as a new vaccine against infection ofmammals with M. tuberculosis is provided. In a further embodiment, animmunogenic composition or vaccine as described herein is effectiveagainst infection in humans. Ag85B-TB10.4 and the IC31® adjuvant induceshigh amounts of polyfunctional CD4⁺T cells and provides significantprotection against M. tuberculosis. Surprisingly, the combination of theAg85B-TB10.4 antigen and the IC31® adjuvant was sensitive to the antigendose. Thus, whereas a standard dose in mice of 5 μg of Ag85B-TB10.4 inIC31® adjuvant did not lead to protection against M. tuberculosis, 0.5μg Ag85B-TB10.4 in IC31® adjuvant induced protection comparable to thatof Bacillus of Calmette and Guerin (BCG).

In an effort to generate an efficient vaccine against infection ofmammals with M. tuberculosis, the combination of the Ag85B-TB10.4 fusionprotein and IC31® adjuvant is used in one or more embodiments.Ag85B-TB10.4 fusion protein has the advantage that it does not includeany of the proteins that are useful for diagnostic purposes such as,e.g., ESAT-6. The absence of ESAT-6 in a vaccine as described hereinwill allow diagnostic tests and a vaccine to be used in parallel sincethe Ag85B-TB10.4 fusion protein does not compromise any of the specificdiagnostic tests. In one embodiment, a vaccine described herein containsan Ag85 protein and TB10.4 as the sole antigens. In a furtherembodiment, a vaccine described herein contains the fusion ofAg85B-TB10.4 as the sole antigen.

In another embodiment, the vaccine excludes (does not contain) dimethyldioctadecyl ammonium bromide (DDA). In yet another embodiment, thevaccine excludes (does not contain) monophosphoryl lipid A (MPL). Inanother embodiment, the vaccine excludes (does not contain) DDA or MPL.In another embodiment, the vaccine contains a mixture a polycationicpeptide and oligodeoxynucleic molecules. In a further embodiment, thevaccine contains a mixture of peptide NH₂-KLKLLLLLKLK-COOH (SEQ ID NO:1)and oligonucleotide 5′-ICI CIC ICI CIC ICI CIC ICI CIC IC-3′ (SEQ IDNO:2)(dIdC)₁₃ (ODN1a; polydeoxyinosinic-deoxycytidylic acid;oligo(dIdC)₁₃) as the sole adjuvant. In a further embodiment, thevaccine contains IC31® adjuvant as the sole adjuvant.

The Ag85B-TB10.4 fusion protein in the IC31® adjuvant constitutes aneffective vaccine against infection in mammals with M. tuberculosis. Ina further embodiment, the vaccine is effective against infection inhumans.

In one embodiment, a vaccine as described is useful as a BCG boostervaccine.

In another embodiment, the Ag85B-TB10.4 fusion protein in the IC31®adjuvant constitutes an effective vaccine against infection with M.tuberculosis. The Ag85B-TB10.4 and IC31® combination induces a highamount of polyfunctional CD4⁺T cells and provides significant protectionagainst M. tuberculosis. Surprisingly, the combination of theAg85B-TB10.4 fusion protein and the IC31® adjuvant is extremelysensitive to the antigen dose. Whereas a dose of 5 μg of Ag85B-TB10.4 inIC31® adjuvant does not lead to significant protection against M.tuberculosis 0.5 μg Ag85B-TB10.4 in IC31® adjuvant induces a strongprotection comparable to that of BCG (Ex. 3). Applicants have identifiedthat Ag85B-TB10.4 is an extraordinarily immunogenic molecule.

In one embodiment, the application is directed to the combination of anAg85B-TB10.4 (Hyvac 4; H4) fusion protein and IC31® adjuvant as a newvaccine against infection with M. tuberculosis. The IC31® adjuvantcomprises cationic peptides and is a TLR9 (toll-like receptor 9)agonist.

A vaccine for mammalian use with a low dose of an antigen comprisingTB10.4 fused to a polypeptide of the antigen 85-complex, e.g., Ag85B inan adjuvant, and methods of immunization against, and treatment of, M.tuberculosis are provided. In a further embodiment, the vaccine is forhuman use.

An immunogenic composition for mammalian use is provided comprising aTB10.4 protein and an Ag85-complex protein which optionally can be fusedtogether or provided as separate proteins wherein the total amount ofprotein is less than about 251g, or less than 10 μg or equal to about0.5 μg per antigen dose. In a further embodiment, the immunogeniccomposition is for human use. In still another embodiment, thecomposition excludes (does not contain) ESAT-6. In one embodiment, acomposition described herein contains an Ag85 protein and TB10.4 as thesole antigens. In a further embodiment, a composition described hereincontains the fusion of Ag85B-TB10.4 as the sole antigen.

In a further embodiment, the composition excludes (does not contain)dimethyl dioctadecyl ammonium bromide (DDA). In another embodiment, thecomposition excludes (does not contain) monophosphoryl lipid A (MPL). Inanother embodiment, the composition excludes (does not contain) DDA orMPL. In another embodiment, the composition contains a mixture apolycationic peptide and oligodeoxynucleic molecules. In a furtherembodiment, the composition contains a mixture of peptideNH₂-KLKLLLLLKLK-COOH (SEQ ID NO:1) and oligonucleotide 5′-ICI CIC ICICIC ICI CIC ICI CIC IC3′ (SEQ ID NO:2)(dIdC)₁₃ (ODN1a;polydeoxyinosinic-deoxycytidylic acid; oligo(dIdC)₁₃) as the soleadjuvant. In a further embodiment, the composition contains IC31®adjuvant as the sole adjuvant.

An immunogenic composition described herein can additionally comprise anadjuvant. In one embodiment, the adjuvant has at least one polycationicpeptide and at least one oligonucleotide, and in a further embodimentthe oligonucleotide is a TLR9 agonist.

In one embodiment, the adjuvant is IC31®. In a further embodiment, theprotein from the Ag85-complex is an Ag85B protein.

Also described is a vaccine for mammalian use comprising an immunogeniccomposition described herein. In a further embodiment, the vaccine isfor human use.

In another embodiment, a method of inducing protection against M.tuberculosis in a mammal is provided, the method comprising introducinginto the mammal an immunogenic composition as described herein. Inanother embodiment, a method of inducing polyfunctional CD4⁺T cells in amammal is provided, the method comprising introducing into the mammal animmunogenic composition as described herein. In another embodiment, amethod of inducing an immune response against M. tuberculosis in amammal is provided, the method comprising introducing into the mammal animmunogenic composition as described above. In further embodiments ofthese methods, the mammal is a human.

Throughout this specification, unless the context requires otherwise,the word “comprise”, or variations thereof such as “comprises” or“comprising”, will be understood to mean the inclusion of a statedelement or integer or group of elements or integers but not theexclusion of any other element or integer or group of elements orintegers. Unless otherwise indicated, the term “about” means ±10% thelimit of weight measurement given.

A. Antigens

In one embodiment, the antigen may comprise a protein of theAg85-complex fused to TB10.4 protein (TB10.4 described in Dietrich, etal., 2005), including, for example, proteins Ag85A, Ag85B or Ag85C ofthe Ag85 complex. The proteins of the antigen 85 complex (85A, 85B, and85C) are encoded by three genes located at different sites in themycobacterial genome, which show extensive cross-reactivity as well ashomology at amino acid and gene levels. The proteins differ slightly inmolecular mass in the 30- to 31-kDa region. The individual components ofthe Ag85 complex (Ag85A, Ag85B and Ag85C) are publicly available (e.g.,from Colorado State University).

In one embodiment, a fusion protein of the TB10.4 protein and anAg85-complex protein may be prepared as described in [Dietrich, et al.,2005]. In another embodiment, a fusion may be prepared by linking theTB10.4 protein to an Ag85-complex protein directly or via a connectinglinker of at least one amino acid. Other methods of preparing the fusionproteins are known conventionally, and are considered to be usefulherein.

In another embodiment, Ag85B-TB10.4 is utilized. In a furtherembodiment, Ag85B-TB10.4 is given in low doses, i.e., doses less thanthose currently used in subunit M. tuberculosis vaccines (e.g., 5 μg to25 μg per dose), to initiate the maximum amount of polyfunctional immunecells, inducing more interferon-γ expression and increased protectionagainst M. tuberculosis. Throughout this specification Ag85B-TB10.4fusion protein is interchangeable with the terms H4 and HyVac4.

In another embodiment the antigen may comprise a protein of the Ag85complex and TB10.4 protein wherein the Ag85 complex protein is not fusedto the TB10.4 protein. In a further embodiment, the protein of the Ag85complex is Ag85B. In one embodiment, the Ag85B-TB10.4 fusion protein isprepared according to Dietrich, et al., 2005.

In still another embodiment, the antigen excludes (does not contain)ESAT-6.

Pro- tein amino acid sequence Ag85A MQLVDRVRGA VTGMSRRLVV GAVGAALVSGLVGAVGGTAT (SEQ AGAFSRPGLP VEYLQVPSPS MGRDIKVQFQ SGGANSPALY ID NO:LLDGLRAQDD FSGWDINTPA FEWYDQSGLS VVMPVGGQSS 3) FYSDWYQPAC GKAGCQTYKWETFLTSELPG WLQANRHVKP TGSAVVGLSM AASSALTLAI YHPQQFVYAG AMSGLLDPSQAMGPTLIGLA MGDAGGYKAS DMWGPKEDPA WQRNDPLLNV GKLIANNTRV WVYCGNGKPSDLGGNNLPAK FLEGFVRTSN IKFQDAYNAG GGHNGVFDFP DSGTHSWEYW GAQLNAMKPDLQRALGATPN TGPAPQGA Ag85B MTDVSRKIRA WGRRLMIGTA AAVVLPGLVG LAGGAATAGA(SEQ FSRPGLPVEY LQVPSPSMGR DIKVQFQSGG NNSPAVYLLD ID NO: GLRAQDDYNGWDINTPAFEW YYQSGLSIVM PVGGQSSFYS 4) DWYSPACGKA GCQTYKWETF LTSELPQWLSANRAVKPTGS AAIGLSMAGS SAMILAAYHP QQFIYAGSLS ALLDPSQGMG PSLIGLAMGDAGGYKAADMW GPSSDPAWER NDPTQQIPKL VANNTRLWVY CGNGTPNELG GANIPAEFLENFVRSSNLKF QDAYNAAGGH NAVENFPPNG THSWEYWGAQ LNAMKGDLQS SLGAG Ag85CMTFFEQVRRL RSAATTLPRR LAIAAMGAVL VYGLVGTFGG (SEQ PATAGAFSRP GLPVEYLQVPSASMGRDIKV QFQGGGPHAV ID NO: YLLDGLRAQD DYNGWDINTP AFEEYYQSGL SVIMPVGGQS5) SFYTDWYQPS QSNGQNYTYK WETFLTREMP AWLQANKGVS PTGNAAVGLS MSGGSALILAAYYPQQFPYA ASLSGFLNPS EGWWPTLIGL AMNDSGGYNA NSMWGPSSDP AWKRNDPMVQIPRLVANNTR IWVYCGNGTP SDLGGDNIPA KFLEGLTLRT NQTFRDTYAA DGGRNGVFNFPPNGTHSWPY WNEQLVAMKA DIQHVLNGAT PPAAPAAPAA TB10. MSQIMYNYPA MLGHAGDMAGYAGTLQSLGA EIAVEQAALQ 4 SAWQGDTGIT YQAWQAQWNQ AMEDLVRAYH AMSSTHEANT (SEQMAMMARDTAE AAKWGG ID NO: 6)

Each protein may be modified by glycosylation, or lipidation (Mowat, etal., 1991; Lustig, et al., 1976). Each protein may be modified by theaddition of prosthetic groups, a purification moiety, or a signalpeptide. Each protein may be modified one or more times or not undergoany modification. Each protein may be modified singly or in combination.Each protein will be characterised by specific amino acids and beencoded by specific nucleic acid sequences. Within the scope of thepresent invention are such sequence and analogues and variants producedby recombinant or synthetic methods wherein such amino acid sequenceshave been modified by substitution, insertion, addition or deletion ofone or more amino acid residues in the recombinant protein whileretaining immunogenicity as confirmed by any one or all of thebiological assays described herein.

Substitutions are preferably “conservative”. These are defined accordingto the following table. Amino acids in the same block in the secondcolumn and preferably in the same line in the third column may besubstituted for each other. The amino acids in the third column areindicated in one-letter code.

ALIPHATIC Non-polar G, A, P I, L, V Polar-uncharged C, S, T, M N, QPolar-charged D, E K, R AROMATIC H, F, W, Y

B. Adjuvants

In one embodiment, the antigen comprises an Ag85 complex protein fusedto TB10.4 protein in an adjuvant. In another embodiment, the antigencomprises an Ag85 complex protein and a TB10.4 protein (i.e., non-fused)in an adjuvant.

Ag85B-TB10.4 is an extraordinary immunogenic molecule which must begiven in low doses to initiate the maximum amount of polyfunctionalimmune cells inducing more interferon-γ expression and increasedprotection against M. tuberculosis. It is exemplified with an adjuvantcomprising a polycationic peptide [e.g., polylysine (KLK peptide) orpolyarginine] and oligodeoxynucleic molecules but is not limited to thisadjuvant.

In a further embodiment, the adjuvant is a mixture of a polycationicpeptide [e.g., polylysine (KLK peptide) or polyarginine] andoligodeoxynucleic molecules [I-ODNs]. An example of a suitable I-ODN foruse with the current invention is oligo dIC, e.g., oligonucleotide5′-ICI CIC ICI CIC ICI CIC ICI CIC IC-3′ (SEQ ID NO:2)(dIdC)₁₃ (ODN1a;polydeoxyinosinic-deoxycytidylic acid; oligo(dIdC)₁₃). I-ODN'sappropriate for use in the embodiments described herein may be found inInternational (PCT) Patent Application Publication Nos.: WO 01/93905 andWO 01/93903, which are hereby incorporated by reference. An example ofKLK peptides suitable for use with the current invention areNH₂-KLKLLLLLKLK-COOH (SEQ ID NO:1) or KLKLLLLLKLK-NH₂ (SEQ ID NO:1).Additional examples of appropriate polycationic peptides areRLRLLLLLRLR-NH₂ (SEQ ID NO:7), RLKLLLLLKLR-NH₂ (SEQ ID NO:8),KFKFFFFFKFK-NH₂ (SEQ ID NO:9), KWKWWWWWKWK-NH₂ (SEQ ID NO:10) orKVKVVVVVKVK-NH₂ (SEQ ID NO:11). Polycationic peptides suitable for usein these embodiments may be found in International (PCT) PatentApplication Publication No. WO/0232451, which is hereby incorporated byreference.

As described herein, the IC31® adjuvant (Intercell AG) comprises amixture of the peptide NH₂-KLKLLLLLKLK-COOH (SEQ ID NO:1) (MultiplePeptide Systems, San Diego, Calif., USA) and the oligonucleotide 5′-ICICIC ICI CIC ICI CIC ICI CIC IC-3′ (SEQ ID NO:2)(dIdC)₁₃ (ODN1a;polydeoxyinosinic-deoxycytidylic acid; oligo(dIdC)₁₃) (Proligo, Boulder,USA).

In one embodiment, a dose of 0.5 μg Ag85B-TB10.4 fusion protein in IC31®adjuvant [100 nmol peptide (NH₂-KLKLLLLLKLK-COOH (SEQ ID NO:1) and 5nmol oligonucleotide 5′-ICI CIC ICI CIC ICI CIC ICI CIC IC-3′ (SEQ IDNO:2)(dIdC)₁₃] induces a strong INF-γ response. However, one of skill inthe art will be able to adjust amounts and concentrations according tothe application. This strong IFN-γ response is in approximately the samerange as 5 μg Ag85B-TB10.4 fusion protein, without IC31® adjuvant. In afurther embodiment, 0.5 μg Ag85B-TB10.4 fusion protein in IC31® adjuvant[100 nmol peptide (NH₂-KLKLLLLLKLK-COOH (SEQ ID NO:1) and 5 nmololigonucleotide 5′-ICI CIC ICI CIC ICI CIC ICI CIC IC-3′ (SEQ IDNO:2)(dIdC)₁₃] is utilized. This provides a strong protection inapproximately the same range as BCG. Without wishing to be bound bytheory, Applicants have determined that this is surprisingly better than5 μg or 15 μg of Ag85B-TB10.4 fusion protein alone (i. e., no IC31®adjuvant), revealing that the preferred dose of Ag85B-TB10.4 fusionprotein alone may be lower.

In one embodiment, less than 5 μg of Ag85B-TB10.4 fusion protein perdose is utilized. In still other embodiments, 1 μg, 0.5 μg, or 0.1 μg ofAg85B-TB10.4 fusion protein is utilized.

As reflected herein, the lowest dose of Ag85B-TB10.4 fusion protein (0.5μg) in IC31® adjuvant gave the highest IFN-γ response after stimulationwith either of the vaccine components (see FIGS. 1A-1D and 2A-2D).Finally, vaccination with Ag85B-TB10.4 fusion protein in IC31® adjuvantinduced two major CD4⁺T cell populations, one expressing IFN-γ, IL-2,and TNF-α, and another expressing IL-2 and TNF-α. Both of these T cellpopulations belong to central memory T cells, and are necessary for longterm memory. Importantly, as seen with the IFN-γ expression measured byELISA, using a dose of 0.5 μg HyVac4 fusion protein in IC31® adjuvantinduced the highest cell numbers within the polyfunctional populationthat expressed IFN-γ, IL-2, and TNF-α.

Applicants have determined that the adjuvants IC31® and cationicliposomes exhibit different sensitivities towards the antigen dose; andthat the preferred antigen dose in IC31® adjuvant is antigen-dependent.

0.5 μg Ag85B-TB10.4 fusion protein in IC31® adjuvant induced significantprotection whereas 5 μg Ag85B-TB10.4 fusion protein in IC31® adjuvantdid not (FIGS. 3A-3B). One of skill in the art will recognize from thisapplication that the appropriate antigen dose in a vaccine depends bothon the antigen and on the adjuvant.

In one embodiment, the composition excludes (does not contain) dimethyldioctadecyl ammonium bromide (DDA). In another embodiment, thecomposition excludes (does not contain) monophosphoryl lipid A (MPL).

The following examples are illustrative of the compositions and methodsof the invention. It will be readily understood by one of skill in theart that the specific conditions described herein can be varied withoutdeparting from the scope of the present invention. It will be furtherunderstood that other compositions not specifically illustrated arewithin the scope of the invention as defined herein.

Examples

The following information is supportive of the examples that follow.

Animals. Studies were performed with 8 to 12 week-old C57BL/6xBalb/c F1female mice, purchased from Taconic, Ejby, Denmark. Infected animalswere housed in cages contained within laminar flow safety enclosures ina BSL-3 facility. The use of mice was in accordance with the regulationsset forward by the Danish Ministry of Justice and Animal ProtectionCommittees and in compliance with EC Directive 86/609 and the US ALACrecommendations for the care and use of Laboratory animals.

Bacteria. M. tuberculosis Erdman were grown at 37° C. onLowenstein-Jensen medium or in suspension in Sauton medium enriched with0.5% sodium pyruvate and 0.5% glucose.

Immunization. Mice were immunized three times at 2-week intervalssubcutaneously on the back with experimental vaccines containing 0.5, 5or 15 μg of Ag85B-TB10.4 fusion protein (H4)/dose, emulsified in IC31®adjuvant in a total volume of 0.2 ml/dose. Doses were 100 nmol peptideand 5 nmol oligonucleotide. All vaccines were formulated using 10 mMTris-HCl/270 mM sorbitol buffer (pH 7.9) as previously described (Olsen,et al., 2001) to obtain a final volume of 0.2 ml/mouse. At the time ofthe first subunit vaccination, one group of mice received a single doseof BCG Danish 1331 (2.5×10⁵ CFU) injected subcutaneously at the base ofthe tail and one group received a saline injection. All groups of micewere challenged 10 weeks after the first vaccination.

Experimental infections. When challenged by the aerosol route, theanimals were infected with approximately 50 CFU of M. tuberculosisErdman/mouse. These mice were sacrificed 6 weeks after challenge.Numbers of bacteria in the spleen or lung were determined by serialthreefold dilutions of individual whole-organ homogenates in duplicateon 7H11 medium (Middlebrook; Sigma-Aldrich). Organs from theBCG-vaccinated animals were grown on medium supplemented with 2 μg of2-thiophene-carboxylic acid hydrazide (TCH)/ml to selectively inhibitthe growth of the residual BCG bacteria in the test organs. Colonieswere counted after 2 to 3 weeks of incubation at 37° C. Bacterial burdenin the lungs was expressed as log₁₀ of the bacterial counts based onvaccination groups of six animals.

Lymphocyte cultures. Lymphocytes from spleens were obtained as describedpreviously (Brandt, et al.). Blood lymphocytes (PBMCs) were purified ona density gradient. Cells pooled from five mice in each experiment werecultured in microtiter wells (96-well plates; Nunc, Roskilde, Denmark)containing 2×10⁵ cells in a volume of 200 μl of RPMI 1640 supplementedwith 5×10⁻⁵ M 2-mercaptoethanol, 1% penicillin-streptomycin, 1 mMglutamine, and 5% (vol/vol) fetal calf serum. Based on previousdose-response investigations, the mycobacterial antigens were all usedat 15 μg/ml or 5 μg/ml, while concanavalin A was used at a concentrationof 1 μg/ml as a positive control for cell viability. All preparationswere tested in cell cultures and found to be nontoxic at theconcentrations used in the present study. Supernatants were harvestedfrom cultures after 72 h of incubation for the investigation of IFN-γ.

IFN-γ enzyme-linked immunosorbent assay (ELISA) Microtiter plates (96wells; Maxisorb; Nunc) were coated with monoclonal hamster anti-murineIFN-γ (Genzyme, Cambridge, Mass.) in PBS at 4° C. Free binding siteswere blocked with 1% (wt/vol) bovine serum albumin-0.05% Tween 20.Culture supernatants were tested in triplicate, and IFN-γ was detectedwith a biotin-labelled rat anti-murine monoclonal antibody (cloneXMG1.2; Pharmingen, San Diego, Calif.). Recombinant IFN-γ (Pharmingen,San Diego, Calif.) was used as a standard.

FACS analysis of lyniphocytes. Intracellular cytokine stainingprocedure: Cells from blood, spleen or lungs of mice were stimulated for1-2 h with 2 μg/ml Ag and subsequently incubated for 6 h with 10 μg/mlbrefeldin A (Sigma-Aldrich, USA) at 37° C. Thereafter, cells were storedovernight at 4° C. The following day, Fc receptors were blocked with 0.5μg/ml anti-CD16/CD32 mAb (BD Pharmingen, USA) for 10 minutes, whereafter the cells were washed in FACS buffer (PBS containing 0.1% sodiumazide and 1% FCS), and stained for surface markers as indicated using0.2 μg/ml anti-CD4 (clone: RM4-5), anti-CD8 (clone: 53-6,7) mAb's. Cellswere then washed in FACS buffer, permeabilized using theCytofix/Cytoperm™ kit (BD Pharmingen, Denmark) according to themanufacturers instructions, and stained intracellularly with 0.2 μg/mlanti-IFN-γ (clone: XMG1.2), anti-TNF-α (clone: MP6-XT22), or anti-IL-2(clone: JES6-5H4) mAb's. After washing, cells were re-suspended informaldehyde solution 4% (w/v) pH 7.0 (Bie & Berntsen, Denmark) andanalysed by flow cytometry on a six-colour BD FACSCanto flow cytometer(BD Biosciences, USA).

Statistical methods. The data obtained were tested by analysis ofvariance. Differences between means were assessed for statisticalsignificance by Tukey's test. A P value of <0.05 was consideredsignificant.

Example 1 Immune Response Induced After Immunization with Ag85B-TB10.4Fusion Protein in IC31® Adjuvant

The immunogenicity of Ag85B-TB10.4 fusion protein delivered in IC31®adjuvant was analyzed, including whether both components of the fusionprotein were recognized by the immune system after immunization.

PBMC's isolated from groups of mice vaccinated with different doses ofH4 in IC31® adjuvant and a saline control group were stimulated witheither 1 or 5 μg/ml of Ag85B, TB10.4 or CFP10 (a Mycobacteriumtuberculosis-specific antigen). After 72 hours the concentration of cellreleased IFN-γ was determined by ELISA. PBMC's were isolated 1 weekafter third vaccination and were pooled from five mice per group. Valuesin FIGS. 1A-1D represent the means of triplicate and SEM's are indicatedby bars.

Groups of mice were immunized with Ag85B-TB10.4 fusion protein in IC31®adjuvant. As negative control, a group of mice received the adjuvantalone (data not shown). To examine antigen dose in IC31® adjuvant, weused 15, 5 and 0.5 μg of Ag85B-TB10.4 fusion protein. One week after thelast injection, mice were bled, and the IFN-γ release was evaluatedafter in vitro stimulation of purified PBMCs with differentconcentrations of Ag85B and TB10.4 proteins (5 μg/ml and 1 μg/ml) (FIG.1A). Immunization with Ag85B-TB10.4 fusion protein in IC31® adjuvantinduced a strong IFN-γ response specific for Ag85B and TB10.4 proteins(FIGS. 1B-1D). Surprisingly, this response was sensitive to the antigenimmunization dose. Thus, the lowest dose of Ag85B-TB10.4 fusion proteinin IC31® adjuvant gave the highest IFN-γ response after stimulation witheither Ag85B (9401±3668 pg/ml IFN-γ) or TB10.4 (4694±3992 pg/ml IFN-γ)(FIG. 1D). Using a dose of 5 μg or 15 μg Ag85B-TB10.4 fusion proteinsignificantly reduced the IFN-γ response against both Ag85B and TB10.4proteins relative to mice vaccinated with 0.5 μg Ag85B-TB10.4 fusionprotein (p<0.001). This was particularly apparent for the highimmunization dose—15 μg Ag85B-TB10.4 fusion protein per immunizationdose (FIG. 1C)—which gave IFN-γ responses that did not differ from theobserved responses in non-vaccinated mice (or in Ag85B-TB10.4 fusionprotein vaccinated mice stimulated in vitro with control antigen CFP10).

The same dose dependency was subsequently repeated in an independentexperiment where the immune responses were analyzed in both blood andspleen (FIGS. 2A-2D). PBMC's (2A)/(2C) and splenocytes (2B)/(2D)isolated from groups of mice immunized with 3 different doses of H4formulated in IC31® adjuvant or a saline control group were stimulatedwith Ag85B (2A)/(2B) or TB10.4 (2C)/(2D) for 72 hours where after IFN-γcytokine secretion was measured by ELISA. The bars represent means of 3individual mice. SEMs are indicated. In FIGS. 2A-2D, a vaccination doseof 0.5 μg H4 gave significantly (p<0.05, one-way ANOVA and Tukey's posttest) higher antigen responses, compared to vaccination doses of 5 μgand 15 μg.

These results show that the lowest dose of 0.5 μg Ag85B-TB10.4 fusionprotein in IC31® adjuvant induced the strongest systemic response of theantigen doses tested.

Example 2 Vaccination with Ag85B-TB10.4 Fusion Protein in IC31®AdjuvantInduces Polyfunctional CD4⁺T Cells

The phenotype of the T cells induced by immunizing with Ag85B-TB10.4fusion protein in IC31® adjuvant was analyzed. In particular, theability of this vaccine to induce polyfunctional (IFN-γ⁺IL-2⁺TNF-α⁺)CD4⁺T cells was determined as these have been shown to correlate withprotective immunity against infections such as Leishmania major and toform the basis for a long lived memory response.

Ag85B and TB10.4 specific T cells are poly-functional. Production ofIFN-γ, TNF-α and IL-2 was assessed following antigenic stimulation ofPBMC's and spleenocytes 2 weeks post-vaccination by flow cytometry.

PBMC's from Ag85B-TB10.4 fusion protein in IC31® adjuvant vaccinatedmice were stimulated in vitro with Ag85B or TB10.4 fusion protein andanalyzed by flow cytometry for expression of CD4, CD8, IFN-γ, TNF-α, andIL-2. The results show that immunizing with Ag85B-TB10.4 fusion proteinin IC31® adjuvant induced two major polyfunctional T cell populations;CD4⁺IFN-γ⁺IL-2⁺TNF-α⁺ and CD4⁺IL-2⁺TNF-α⁺T cells. This was seen forAg85B and TB10.4 specific T cells. Interestingly, as observed in FIG. 1,there is a higher response in the group immunized with 0.5 μg HyVac4 inIC31® adjuvant compared to the group immunized with 5 μg HyVac4 fusionprotein in IC31® adjuvant, and that the major difference was that thegroup being vaccinated with only 0.5 μg showed significantly morepolyfunctional T cells. Taken together, immunizing with Ag85B-TB10.4fusion protein in IC31® adjuvant induced poly-functional CD4⁺T cells andconfirmed that lowering the amount of Ag85B-TB10.4 fusion proteinincreased the immunogenicity of the vaccine in terms of not only IFN-γexpression but also the number of polyfunctional T cells.

Example 3 Protective Efficacy of Ag85B-TB10.4 Fusion Protein and IC31®Adjuvant in a Mouse M. tuberculosis Infection Model

The protective efficacy of Ag85B-TB10.4 fusion protein in IC31® adjuvantwas examined, including whether the dose dependency regarding theimmunogenicity of the vaccine was also reflected in the protectiveefficacy of the vaccine.

In two independent experiments (A and B) groups of mice were vaccinatedwith three different doses of H4 formulated in IC31® adjuvant andcompared to saline and BCG-vaccinated controls.

Mice were vaccinated three times at two weeks interval with Ag85B-TB10.4fusion protein in IC31® adjuvant. As a positive control for protection,a group of mice were immunized once with BCG.

Ten weeks after the first vaccination, the mice were challenged by theaerosol route with virulent M. tuberculosis Erdman. Six weeks postchallenge, the mice were sacrificed and the numbers [bacterial burden(CFU)] were determined in the lungs. As observed with the immunogenicityof the vaccines, the lowest Ag85B-TB10.4 fusion protein immunizationdose induced the highest protection. Thus, mice vaccinated with 0.5 μgAg85B-TB10.4 fusion protein in IC31® adjuvant contained a bacterialnumber of 5.0±0.2 Log₁₀ CFU in the lungs. This was equal to the numbersobserved in BCG vaccinated mice (4.90±0.35 Log₁₀ CFU), but significantlylower (p<0.001) compared to the bacterial numbers in non-vaccinated mice(5.83±0.12 Log₁₀ CFU) (FIG. 3A). In contrast, the bacterial numbers inmice vaccinated with 5 μg or 15 μg of Ag85B-TB10.4 fusion protein inIC31® adjuvant, were not significantly different from the levels foundin the lungs of non-vaccinated mice (FIG. 3A). Repeating the experimentled to the same conclusion although the overall bacterial numbers wereslightly lower in all the groups (FIG. 3B). Thus the ability of thevaccine, Ag85B-TB10.4 fusion protein in IC31® adjuvant, to induceprotection against M. tuberculosis correlated with the immunogenicity ofthe vaccine, in terms of IFN-γ production and the number ofpolyfunctional T cells, and was highest when the lowest antigen dose wasused.

In both experiments, data are presented as mean values from six animalsper group and standard errors of the means are indicated by bars.Statistical comparisons among the vaccination groups were done byone-way ANOVA and Tukey's post test. Significant differences are onlyshown for selected groups. ***: p<0.001, *: p<0.05.

The surprising in vivo results from these well recognized M.tuberculosis animal models, supports the use of the immunogeniccompositions of the current invention as a M. tuberculosis vaccine inhumans.

Example 4 Protective Efficacy of Ag85B-TB10.4 Fusion Protein and IC31®Adjuvant in a Clinical Trial

In human clinical trials subjects will be vaccinated with less thanabout 5 μg to 25 μg of Ag85B-TB10.4 in IC31® adjuvant. This low dose ofAg85B-TB10.4 is in stark contrast with other subunit M. tuberculosisvaccines currently in clinical trials. For example, 40 μg of MTB72F inAS02A per dose (Leroux-Roels, et al., 2005) and 50 μg of Ag85B-ESAT-6 inIC31® adjuvant per dose (clinical data not published yet).

PUBLICATIONS

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Lingnau, A. von Gabain, C. S. Andersen, K. S. Korsholm, and P. Andersen.2006.

Protective immunity to tuberculosis with Ag85B-ESAT-6 in a syntheticcationic adjuvant system IC31. Vaccine 24:5452-5460.

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Ravn,    and P. Andersen. 2004. Specific T-cell eptopes for imunoassay-based    diagnosis of Mycobacterium tuberculosis infection. J Clin Microbiol    42:2379-2387.-   6. Darrah, P. A., D. T. Patel, P. M. De Luca, R. W. Lindsay, D. F.    Davey, B. J. Flynn, S. T. Hoff, P. Andersen, S. G. Reed, S. L.    Morris, M. Roederer, and R. A. Seder. 2007. Multifunctional TH1    cells define a correlate of vaccine-mediated protection against    Leishmania major. Nat Med 13:843-850.-   7. Dietrich, J., C. Andersen, R. Rappuoli, T. M. Doherty, C. G.    Jensen, and P. Andersen. 2006. Mucosal administration of    Ag85B-ESAT-6 protects against infection with Mycobacterium    tuberculosis and boosts prior bacillus Calmette-Guerin immunity. J    Immunol 177:6353-6360.-   8. Dietrich, J., C. Aagaard, R. Leah, A. W. Olsen, A. Stryhn, T. M.    Doherty, and P. Andersen. 2005. Exchanging ESAT6 with TB10.4 in an    Ag85B fusion molecule-based tuberculosis subunit vaccine: efficient    protection and ESAT6-based sensitive monitoring of vaccine efficacy.    J Immunol 174:6332-6339.-   9. Horwitz, M. A., G. Harth, B. J. Dillon, and S.    Maslesa-Galic. 2000. Recombinant bacillus calmette-guerin (BCG)    vaccines expressing the Mycobacterium tuberculosis 30-kDa major    secretory protein induce greater protective immunity against    tuberculosis than conventional BCG vaccines in a highly susceptible    animal model. Proc Natl Acad Sci USA 97:13853-13858.-   10. Lalvani, A., A. A. Pathan, H. McShane, R. J. Wilkinson, M.    Latif, C. P. Conlon, G. Pasvol, and A. V. Hill. 2001. Rapid    detection of Mycobacterium tuberculosis infection by enumeration of    antigen-specific T cells. Am J Respir Crit Care Med 163:824-828.-   11. Lustig J V, Rieger H L, Kraft S C, Hunter R, Rotlhberg R M.    1976, Cell Iminunol 24(1):164-7.-   12. McShane, H., A. A. Pathan, C. R. Sander, S. M. Keating, S. C.    Gilbert, K. Huygen, H. A. Fletcher, and A. V. Hill. 2004.    Recombinant modified vaccinia virus Ankara expressing antigen 85A    boosts BCG-primed and naturally acquired antimycobacterial immunity    in humans. Nat Med 10: 1240-1244.-   13 Mowat A M, Donachie A M, Reid G, Jarrett O. 1991, Immunology    72(3):317-22-   14. Olsen, A. W., L. A. van Pinxteren, L. M. Okkels, P. B.    Rasmussen, and P. Andersen, 2001. Protection of mice with a    tuberculosis subunit vaccine based on a fusion protein of antigen    85b and esat-6. Infection and Immunity 69:2773-2778.-   15. Ravn, P., A. Demissie, T. Eguale, H. Wondwosson, D. Lein, H.    Amoudy, A. S. Mustafa, A. K. Jensen, A. Holm, I. Rosenkrands, F.    Oftung, J. Olobo, C. F. von-Reyn, and P. Andersen. 1999. Human T    cell responses to the ESAT-6 antigen from Mycobacterium    tuberculosis. J. Infect. Dis. 179:637-645.-   16. Skeiky, Y. A., M. R. Alderson, P. J. Ovendale, J. A.    Guderian, L. Brandt, D. C. Dillon, A. Campos-Neto, Y. Lobet, W.    Dalemans, I. M. Orme, and S. G. Reed. 2004. Differential immune    responses and protective efficacy induced by components of a    tuberculosis polyprotein vaccine, Mtb72F, delivered as naked DNA or    recombinant protein. J Immunol 172:7618-7628.-   17. Stockinger, B., C. Bourgeois, and G. Kassiotis. 2006. CD4+    memory T cells: functional differentiation and homeostasis. Immunol    Rev 211:39-48.-   18. Weinreich Olsen, A., L. A. van Pinxteren, L. Meng Okkels, P.    Birk Rasmussen, and P. Andersen. 2001. Protection of mice with a    tuberculosis subunit vaccine based on a fusion protein of antigen    85b and esat-6. Infect Immun 69:2773-2778.-   19. Wu, C. Y., J. R. Kirman, M. J. Rotte, D. F. Davey, S. P.    Perfetto, E. G. Rhee, B. L. Freidag, B. J. Hill, D. C. Douek,    and R. A. Seder. 2002. Distinct lineages of T(H)1 cells have    differential capacities for memory cell generation in vivo. Nature    immunology 3:852-858.-   20. 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All publications listed in this specification are incorporated herein byreference. While the invention has been described with reference toparticular embodiments, it will be appreciated that modifications can bemade without departing from the spirit of the invention. Suchmodifications are intended to fall within the scope of the appendedclaims.

1. An immunogenic composition comprising a TB10.4 protein and anAg85-complex protein, wherein the total amount of the TB10.4 protein andthe Ag85-complex protein is less than about 25 μg, provided that thecomposition excludes dimethyl dioctadecyl ammonium bromide (DDA).
 2. Animmunogenic composition according to claim 1, wherein the TB10.4 proteinand the Ag85-complex protein are present as a fusion protein.
 3. Animmunogenic composition according to claim 1, further comprising anadjuvant.
 4. An immunogenic composition comprising a TB10.4 protein, anAg85-complex protein, and an adjuvant comprising at least onepolycationic peptide and at least one oligonucleotide, wherein the totalamount of the TB10.4 protein and the Ag85-complex protein is less thanabout 25 μg.
 5. The immunogenic composition according to claim 4,wherein the Ag85-complex protein is an Ag85B protein.
 6. The immunogeniccomposition according to claim 4, wherein the fusion protein is presentin an amount of less than about 10 μg.
 7. The immunogenic compositionaccording to claim 4, wherein the fusion protein is present in an amountequal to about 0.5 μg.
 8. The immunogenic composition according to claim4, wherein the at least one oligonucleotide is a TLR9 agonist.
 9. Theimmunogenic composition according to claim 4, wherein the adjuvant is amixture of peptide NH₂-KLKLLLLLKLK-COOH (SEQ ID NO:1) andoligonucleotide 5′-ICI CIC ICI CIC ICI CIC ICI CIC IC-3′ (SEQ ID NO:2).10. The immunogenic composition according to claim 9, wherein theadjuvant is IC31® adjuvant.
 11. A vaccine comprising a TB10.4 protein,an Ag85-complex protein, and an adjuvant comprising at least onepolycationic peptide and at least one oligonucleotide, wherein the totalamount of the TB10.4 protein and the Ag85-complex protein is less thanabout 25 μg.
 12. The vaccine according to claim 11, wherein theAg85-complex protein is an Ag85B protein.
 13. The vaccine according toclaim 11, wherein the total amount of TB10.4 protein and theAg85-complex protein less than about 10 μg.
 14. The vaccine according toclaim 13, wherein the total amount of TB10.4 protein and theAg85-complex protein equal to about 0.5 μg.
 15. The vaccine according toclaim 11, wherein the at least one oligonucleotide is a TLR9 agonist.16. The vaccine according to claim 11, wherein the adjuvant is a mixtureof peptide NH₂-KLKLLLLLKLK-COOH (SEQ ID NO:1) and oligonucleotide 5′-ICICIC ICI CIC ICI CIC ICI CIC IC-3′ (SEQ ID NO:2).
 17. The vaccineaccording to claim 16, wherein the adjuvant is IC31® adjuvant.
 18. Amethod of inducing protection against M. tuberculosis in a mammal, themethod comprising introducing into the mammal an immunogenic compositionaccording to claim
 1. 19. A method of inducing protection against M.tuberculosis in a mammal, the method comprising introducing into themammal an immunogenic composition according to claim
 4. 20. A method ofinducing an immune response against M. tuberculosis in a mammal, themethod comprising introducing into the mammal an immunogenic compositionaccording to claim
 1. 21. The method according to claim 20, wherein saidmammal is a human.